We propose to investigate the 3D structure, catalytic mechanism, and method of regulation of soluble methane monooxygenase (MMO). MMO catalyzes the first step in the oxidation of CH4 to CO2 by methanotrophic bacteria. In this way, the atmospheric egress of nearly all of the enormous quantify of CH4 (a greenhouse gas with 20 times the potency of C02) generated by anaerobic bacteria is prevented. MMO also adventitiously catalyzes the oxidation of many other hydrocarbons leading to applications in synthesis and in biodegradation. We have purified MMO from the type II methanotroph, Methylosinus trichosporius OB3b; it is composed of 3 proteins termed hydroxylase (MMOH), reductase (MMOR), and "B" (MMOB). Our crystal structure of MMOH shows it to contain a novel active site bis-mu-hydroxo bridged dinuclear iron cluster which is essential for catalysis Spectroscopic studies (including optical, EPR, Mossbauer, EXAFS, ENDOR, resonance Raman, fluorescence, NMR, MCD, and CD), turnover of diagnostic substrates, and transient kinetics are also being used to investigate the structure and molecular mechanism. We hypothesize that O2 leads to the [Fe(II)-Fe(II)]state of the cluster resulting in heterolytic O-O bond cleavage to form a reactive intermediate, formally an [oxo-Fe(iv)-Fe(IV)]species, which attack hydrocarbons with the intermediate formation of a substrate radical or cation. Transient kinetic studies from the project have revealed 2 stable and 7 transient intermediates in the reaction cycle. Spectroscopic studies show that one intermediate, compound Q, contains a bis-mu-oxo-Fe(IV) cluster which reacts directly with CH4 to give CH3OH in support of the mechanistic proposal. Q is the first intermediate to be isolated in an oxygenase that can attach unactivated hydrocarbons. Ongoing studies suggest that MMOR and MMOB regulate catalysis by increasing the rate of Q formation. MMOB mutants have been purified. MMOB mutants have been purified that allow the rate of each step in the catalytic cycle to be individually regulated. Proposed studies focus on mapping the MMO ternary protein complex and using the MMOB mutants to trap and characterize the chemistry of reaction cycle intermediates. This work should yield a fundamental understanding of a new type of O2 activation chemistry, insight into the structure and reactivity of the novel compound Q, and guidance in the design of catalysts for oxidation of abundant hydrocarbons. It is also likely to contribute to our understanding of a new role for protein-protein interactions in biological regulatory processes.